MGDF, or megakaryocyte growth and development factor, is a recently cloned cytokine that appears to be the major regulator of circulating platelet levels. See Bartley, T. D. et al., Cell 77: 1117-1124 (1994); Lok, S. et al., Nature 369: 565-568 (1994); de Sauvage, F. J. et al., Nature 369: 533-538 (1994); Miyazake, H. et al., Exp. Hematol. 22: 838 (1994); and Kuter, D. J. et al., PNAS USA, 91: 11104-11108 (1994). MGDF as described in Bartley, T. D. et al., Cell 77: 1117-1124 (1994), is also referred to as thrombopoietin (TPO), mpl ligand, and megapoietin. Herein, the term "mpl ligand" will be used generically to refer to all polypeptides that activate the mpl receptor, including TPO and MGDF. The mpl receptor is a cell surface protein that, upon activation, leads to production and/or development of megakaryocytes and platelets. See WO 92/07074.
"Mpl ligand analogs" are polypeptides that differ from native sequences in a way that affects the number, location or type of carbohydrate linkage sites. Such polypeptides are one aspect of the present invention. Mature native human mpl ligand is a protein having 332 amino acids in total. The sequence of this protein (attached to a 21-amino acid long leader sequence) and the corresponding cDNA are shown in FIGS. 1A-1C herein (SEQ. ID NOs.: 1 and 2).
Recombinant mpl ligand produced in both Chinese Hamster Ovary (CHO) and E. coli cells has been demonstrated to have a biological activity of specifically stimulating or increasing megakaryocytes and/or platelets in vivo in mice, rats and monkeys. See e.g., Hunt, P. et al., Blood 84(10): 390A (1994). Human mpl ligand molecules that have been truncated (as compared to the 332 amino acid protein encoded by the cDNA in humans) so that they extend at least 151 amino acids, starting from amino acid position 22 in FIG. 1A, retain biological activity in vivo. FIGS. 2A-2B (SEQ. ID NOs.: 3 and 4) shows one example of a truncated mpl ligand molecule which, in mature form, has 174 amino acids and has biological activity. In FIG. 2A, the 174 amino acid long protein is shown attached to a 21 amino acid N-terminal leader sequence. This molecule was used to create some of the mpl ligand analogs in the examples section below. Other analogs are based on amino acids 1-199, 1-191, and 1-183 of FIG. 1A-1C. It is also possible to remove up to the first six amino acids at the N-terminus of the mature human sequence mpl ligand protein and retain biological activity. Therefore, it appears that biological activity is retained within amino acids 7 to 151 (inclusive) of the mature amino acid sequence of human mpl ligand shown in FIG. 1A-1C.
In general, many cell surface and secretory proteins produced by eukaryotic cells are modified with one or more oligosaccharide groups. This modification, referred to as glycosylation, can dramatically affect the physical properties of proteins and can also be important in protein stability, secretion, and subcellular localization. Proper glycosylation can be essential for biological activity. In fact, some genes from eukaryotic organisms, when expressed in bacteria (e.g., E. coli) which lack cellular processes for glycosylating proteins, yield proteins that are recovered with little or no activity by virtue of their lack of glycosylation.
Glycosylation occurs at specific locations or sites along the polypeptide backbone and is usually of two types: O-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues while N-linked oligosaccharides (chains) are attached to asparagine (Asn) residues when they are part of the sequence Asn-X-Ser/Thr, where x can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids not counting proline. The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type are different. One type of sugar that is commonly found on both is N-acetylneuraminic acid (hereafter referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, by virtue of its negative charge, may confer acidic properties to the glycoprotein.
As used herein glycosylation "sites" are amino acid residues that are structurally able to link to glycosyl residues, although such sites may or may not be actually linked to a glycosyl residue. As noted above, O-linked sites are either Ser or Thr residues, whereas N-linked sites are either Asn-X-Ser or Asn-X-Thr, where X is defined as any amino acid other than Pro (preferably one of the 19 naturally-occurring amino acids, excluding Pro). Whether a given site is glycosylated with a glycosyl chain is determined by the host cell in which the molecule is expressed, the amino acids neighboring the site, and other factors.
As used herein, the number of "chains" attached to a given mpl ligand analog will be the average number of carbohydrate (i.e., glycosyl) chains attached to a given mpl ligand molecule expressed by a particular host cell. Notably, the glycosylation sites for natural and corresponding recombinant mpl ligand will generally be the same, whereas the number of chains will possibly vary depending upon whether the particular host cell used for recombinant expression attaches glycosyl chains to the same sites or not, as compared to the natural source. Herein, whenever a comparison is made between recombinant and natural mpl ligand analogs, the same number of amino acids will be compared, regardless of whether the natural source actually produces an mpl ligand molecule having that length. Thus, "natural" refers to the sequence employed in a particular species (such as human) rather than the length of the molecule actually expressed in such natural source.
Naturally occurring mpl ligand is a glycosylated molecule. The glycosylation pattern of natural mpl ligand is related to two key domains that have been found in mpl ligand. The sequence of the first approximately 151 amino acids of mature human mpl ligand, corresponding to an active portion of the molecule, bears notable homology to erythropoietin (EPO), a cytokine capable of stimulating production of erythrocytes, and is referred to as the "EPO-like" domain of human mpl ligand. The remaining amino acids of the mature protein make up a so-called "N-linked carbohydrate" domain, since they include most if not all of the natural sites for N-linked glycosylation. In human mpl ligand, there are six N-linked glycosylation sites all contained in the N-linked glycosylation domain. Both domains contain O-linked glycosylation sites. There are an estimated 12-14 O-linked glycosylation chains in the molecule. Experimental evidence with human mpl ligand DNA expressed recombinantly in CHO cells reveals that in the EPO-like domain at least two O-linked sites are glycosylated, at positions 1 (Ser) and 37 (Thr).
Glycoproteins such as mpl ligand can be separated into different charged forms using techniques such as isoelectric focusing (IEF). For example, several parties have reported IEF studies of crude and partially purified erythropoietin preparations (Lukowsky et al., J. Biochem. 50: 909 (1972); Shelton et al., Biochem. Med. 12: 45 (1975); Fuhr et al., Biochem. Biophys. Res. Comm. 98: 930 (1981)).
In spite of the above information on glycosylation of mpl ligand molecules, there remains a need to obtain mpl ligand molecules having a different glycosylation pattern and which retain or have improved biological activity.
Accordingly, it is an object of the present invention to provide novel glycosylated mpl ligand molecules, referred to as mpl ligand analogs. It is a further object of this invention to provide pharmaceutical compositions containing such molecules and methods of treating conditions treatable by mpl ligand with the mpl ligand analogs of this invention.